This research proposal is an experimental project that will develop an optimal formulation method for gene therapy in the lungs using non-viral gene carriers. Cystic Fibrosis (CF) is a disease caused by a single-gene defect, which leads to impaired function of the CFTR protein. Although this gene was isolated more than 20 years ago, the development of safe and effective delivery strategies have been difficult to realize. A major limitation in the development of non-viral gene carriers has been the lack of basic understanding of the extracellular and intracellular barriers. For example, it was previously shown that the gene transfer efficiency of gene carriers exposed to mucus lining was dramatically reduced, when compared to mucus-depleted tissues. This is because mucus acts as the primary barrier in the CF lungs, and efficiently removes gene carriers prior to reaching the underlying airway epithelium. To address this, we will determine the rate limiting barriers through human CF mucus and human bronchial epithelial cells for efficient delivery of gene carriers. The approach to this range of studies will be one of systematic building from simple to more complex systems, with an emphasis on quantitative studies at each step. The specific aims of this proposal are: (1) to formulate methods that produce gene carriers that are stable in CF mucus. (2) to determine and quantify gene carrier transport through human CF mucus; (3) to determine intracellular barriers to efficient gene carrier transport to the nucleus in human bronchial epithelial cells; and (4) to determine in vivo gene transfer efficiencies and nasal potential differences (NPD) in mice. We hypothesize that the identification of important extra- and intracellular barriers will guide the rational modification of the non-viral gene carriers for successful CF gene therapy.